Method and apparatus for viable and nonviable prokaryotic and eukaryotic cell quantitation

ABSTRACT

A rapid method for the quantitation of various live cell types is described. This new cell fluorescence method correlates with other methods of enumerating cells such as the standard plate count, the methylene blue method and the slide viability technique. The method is particularly useful in several applications such as: a) quantitating bacteria in milk, yogurt, cheese, meat and other foods, b) quantitating yeast cells in brewing, fermentation and bread making, c) quantitating mammalian cells in research, food and clinical settings. The method is especially useful when both total and viable cell counts are required such as in the brewing industry. The method can also be employed to determine the metabolic activity of cells in a sample. The apparatus, device, and/or system used for cell quantitation is also disclosed.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation application of U.S. patentapplication Ser. No. 12/340,044, filed Dec. 19, 2008; which is adivisional application of U.S. patent application Ser. No. 11/144,244,filed Jun. 2, 2005, now U.S. Pat. No. 7,527,924; which is a divisionalapplication of U.S. patent application Ser. No. 09/912,266, filed Jul.24, 2001, now abandoned; which claims priority to U.S. ProvisionalApplication No. 60/220,298, filed Jul. 24, 2000.

BACKGROUND OF THE INVENTION

1. Technical Field

The present invention relates to methods for the rapid quantitation ofboth viable and nonviable cells. More specifically, the inventioninvolves incubating cells with a metabolically activated visiblefluorescent dye and measuring the fluorescence generated by viablecells. Total cell populations (viable+nonviable) are separatelydetermined by measuring the native UV fluorescence of the cells. The twofluorescence readings are directly related to the number of viable andnonviable cells. This permits the user to determine the percentviability of a mixed population of live and dead cells.

2. Description of the Related Art

The ability to quantify living cells is vitally important to the food,beverage, pharmaceutical, environmental, manufacturing and clinicalindustries. Several methods are currently employed by these industriesto quantify prokaryotic and eukaryotic cells. These methods include, butare not limited to, the standard plate count, dye reduction andexclusion methods, electrometric techniques, microscopy, flow cytometry,bioluminescence and turbidity.

The standard plate count permits the quantitation of living cells (orclumps of cells) also known as colony forming units (cfu) when the cellsare grown on the appropriate medium under optimal growth conditions(Microbial Ecology, Atlas, R. M. and Bartha, R., Addison Wesley,Longman, New York, 1998). Current standards of viable organism countsare often based on the standard plate count, particularly in the foodindustry. However, colony counts are difficult to interpret sincebacteria often clump or form chains that can give rise to significantlyinaccurate estimations of the total number of viable organisms in asample. Also, bacteria, for example, can be in a “metabolically damaged”state and not form countable colonies on a given medium. This problem ismore severe when selective media are used. Thus, the standard platecount does not provide a definitive count of viable cells in a sample,which may be very important for certain purposes. Given these factors,such testing also requires skilled technicians who can distinguishindividual colony forming units and who can aid in selecting appropriategrowth medium. Moreover, the technique is not useful when rapiddetermination of cell counts is required, since it often requires over24 hours to obtain results.

Other tests, such as dye reduction tests, rely on the ability of cellsto oxidize or reduce a particular dye (Harrington, 1998). Such methodsare used to measure the activity of metabolically active organismsrather than provide a measure of the total number of viable cells in asample. Dyes, such as methylene blue, coupled with microscopic counting,are routinely employed to determine the relative number ofmicroorganisms. The technique is widely employed but neverthelesssuffers from factors that must be held constant during the assay, e.g.,medium used, chemical conditions, temperature and the types of cellsbeing examined. Also, dye reduction tests that incorporate microscopiccounting techniques require trained technical personnel and often dependon subjective interpretations.

Dye exclusion methods of cell quantitation depend on the living cellshaving the ability to pump the dye out of the cell and into thesurrounding fluid medium. While the dye may enter the interior of bothliving and dead cells, dead cells are not capable of actively pumpingthe dye out under the conditions normally used. Dye exclusion iscommonly employed to enumerate animal, fungal and yeast cells. It is amethod requiring skill, correct timing and proper choice of dye. It isnot applicable to certain microbes and it yields incorrect viable countswith stressed cells.

An accurate estimation of the number of viable yeast cells in a samplecan be obtained by the slide viability technique (Gilliland, 1959). Theyeast cells are suspended in a growth medium containing 6% gelatin andthe suspension is placed in a hemacytometer slide. The cell suspensionis incubated for approximately 20 hours and the numbers of microcolonies are counted. Cells that form micro colonies are viable and deadcells remain as single cells. This technique is considered by thebrewing industry to be the most definitive test for counting the numberof viable yeast cells. Unfortunately, the long incubation time makes itunacceptable as a routine method.

Microscopic techniques typically involve counting a dilution of cells ona calibrated microscopic grid, such as a hemacytometer. A recentimprovement in this technique is the direct epifluorescent filtertechnique (DEFT) (Pettipher et al, 1989). In this technique, samples arefiltered through a membrane filter that traps the cells to be counted. Afluorescent dye is attached to the cells, which are illuminated withultraviolet light and counted. Unfortunately, the technique requires theuse of an expensive microscope and a trained individual or an expensiveautomated system (Pettipher et al., 1989).

Yet other methods of quantitation use flow cytometry, which involves thedifferential fluorescent staining of cells suspended in a relativelyclear fluid stream of low viscosity. The cell suspension is mixed withthe fluorescent dye and illuminated in a flow cell by a laser or otherlight source. The labeled cells are automatically detected with the useof a fluorescence detector focused on the cells (Brailsford and Gatley,1993 and Pinder et al., 1993). The technique requires, and is limitedby, expensive equipment. Some flow cytometric devices have been used bythe food and dairy industry, but their application has been limited bythe high cost of instrumentation.

Bioluminescence has been routinely employed in the food sanitationindustry to detect and quantify viable organisms and cells. The methodinvolves the use of luciferin-luciferase to detect the presence of ATP(Harrington, 1998 and Griffith et al., 1994). When used to quantifycells, the technique depends on the assumption that there is a constantamount of ATP in a living cell. ATP levels vary in a single cell overmore than two orders of magnitude, making this method a relativelyinaccurate technique for the enumeration of viable organisms in asample.

Turbidity of a liquid sample can also be measured as an indication ofthe concentration of cells due to the light scattering and absorbingqualities of suspended cells (Harrington, 1998). The method is old butit is still employed to estimate the bacterial concentration in asample. The method is rapid and simple but is highly inaccurate sinceall cells, particles and substances, including non-living particulatematter, interfere with the interpretation of the results.

The present invention for the quantitation of both viable and nonviablecells is designed to overcome at least five problems that have beenidentified within the field. First, the new technology circumvents theneed for training personnel in how to plate, grow and count viable cellsfrom colonies on agar plates. It also eliminates nearly all training andmaintenance costs associated with most of the other methods. Second, theinvention substantially decreases the time needed to determineconcentrations of cells such as yeast and bacteria. Under currentmethodologies, quantification requires from 24-72 hours (plate count andenrichment cultures), while the present invention permits accuratequantitation in less than 15 minutes. The methylene blue test is rapid;however, the accuracy is unacceptable for cultures that are less than90% viable. The slide viability test is accurate for large viabilityranges but the time required for results is not suitable for routineuse. Third, the new test is accurate over wide ranges of viability andhas precision similar to the slide viability test. Fourth, the instantinvention offers substantial cost savings over existing methods of cellquantitation. Fifth, the invention permits the simultaneousdetermination of both viable and total cells in a sample. This allowsthe user to accurately establish the percent viability of a cell sample(the number of viable cells to total cells). Percent viability is acrucial measurement in many industries such as the dairy and beerbrewing industries and is currently carried out by the methylene bluetest.

BRIEF SUMMARY

The present invention generally provides methods, kits, and devices fordetecting and quantitating the number and/or percentage of viable cellsin a sample. In one aspect the invention provides a method fordetermining the percent viability of cells in a sample, comprisingproviding a sample containing said cells, detecting the total cellcount, contacting said cells with molecule or dye that is detectablyaltered by enzymatic activity of a viable cell, detecting enzymaticallyaltered dye or molecule, thereby detecting the number of viable cellsand comparing the number of total cells with the number of viable cellsthereby determining the percent viability.

In another aspect, a method for detecting viable cells is provided thatcomprises providing a sample containing cells, contacting said samplewith a dye that diffuses or is transported into said cells and whereinsaid dye is detectably altered by enzymatic activity of a viable cell,thereby detecting viable cells in a sample.

Yet additional aspects of the present invention include methods forquantitating viable cells in a sample, comprising providing a samplecontaining said cells, contacting said cells with molecule or dye thatis detectably altered by enzymatic activity of a viable cell, detectingenzymatically altered dye or molecule, thereby detecting the number ofviable cells in said sample and obtaining a value therefrom andcorrelating the detected viable cell value with a standard value,thereby quantitating the viable cells in said sample.

Further aspects include methods for quantitating total and live cells ina sample, comprising measuring total fluorescence of cells in a sampleand comparing to a standard value, thereby quantitating total cells insaid sample; contacting a sample with a fluorescent dye that ismetabolically altered by live cells; said dye having fluorescenceproperties that are measurably altered when modified by live cells,detecting the metabolic alteration of the dye thereby obtaining ameasurement value and comparing said value to a standard value, therebyquantitating live cells in said sample.

Still other aspects of the present invention include methods formeasuring the number of total and live yeast, bacteria or other cells ina sample, comprising measuring the native fluorescence of cells insuspension, contacting said cells with a dye that penetrates into theinterior of yeast or bacteria and is metabolically modified to ameasurable parameter by live cells, measuring the total fluorescence andfluorescence properties provided by the metabolic alteration of saidsample and correlating said fluorescence to the number of total and livecells in said sample or a fraction of the sample and determining thepercent viability of said sample.

In certain embodiments the cells may be of any origin such as bacteria,yeast, or mammalian. In related embodiments the total cell count isdetermined by a method selected from the group consisting of native UVabsorption, turbidity testing, hemacytometer measurements, fluorescence,and dye exclusion.

In yet other embodiments, the enzymatic activity that alters the dye ormolecule is esterase activity. In further embodiments the enzymaticallyaltered dye or molecule is fluorescein diacetate or OREGON GREEN™.

Other embodiments include measurement by a device, such as by afluorometer.

The invention also provides kits for quantifying yeast or bacteria,comprising a cell suspension solution, a cell penetrating dye, andinstructions for detecting dye that correlates to hemacytometer counts,plate counts or other methods of counting viable cells.

In certain embodiments the kit includes a dye that is enzymatically anddetectably altered following penetration of viable cells.

In certain aspects a kit for quantifying yeast or bacteria or mammaliancells is provided, comprising: a first container containing a firstsolution, a second solution containing a compound that penetrates cellmembranes and is metabolized to a fluorescent dye or other detectabledye that is measurable, and instructions for using the same.

In other embodiments kits of the invention further comprise a means formixing said first solution with a sample containing an unknown number ofliving cells and nonliving cells, means for concentrating the cells fromthe mixture of said first solution with said sample and removing solidsfrom the remainder of said mixture, and measuring native fluorescence ofcells in said solution.

In still yet other embodiments the kits may further comprise a means formixing said second solution with said cells to form a second mixture,and means for illuminating the mixture of said second solution with saidcells with excitation light and measuring fluorescence emitted by saidmixture, and thereby determining the amount of metabolically modifieddye present in the cells that is proportional to the number of viablecells in said second solution.

In other embodiments the kits may further comprise a third solutioncontaining a compound or compounds that increase the rate of uptake ofdye into cells or speed up the rate of conversion of the detectablefluorescent form of the dye inside said cells in second solution.

In another embodiment, a device comprising solid fluorescent materialconsisting of an adaptor and a compound that can be used to calibratethe instrumentation used for detecting fluorescence in the cells isdisclosed.

These and other aspects of the present invention will become evidentupon reference to the following detailed description and attacheddrawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an example of solid calibration standards for an ultravioletand visible wavelength fluorometer.

FIG. 2 is a correlation chart of comparable Easy Count readings of totalcell counts to total cell counts as determined by methlylene blue.

FIG. 3 is a correlation chart of comparable Easy Count readings ofviable cell counts to total cell counts as determined by methlyleneblue.

FIG. 4 is a plot of regression analysis demonstrating the relationshipbetween the inventive method and those determined by methylene blue fortotal cell count.

FIG. 5 is a plot of regression analysis demonstrating the relationshipbetween the inventive method and those determined by methylene blue forviable cell counts.

FIG. 6 is a linear correlation plot of hemacytometer counts vs. EasyCount Readings. Data points are the mean of three samples. The linearrelationship is significant P<0.0001.

FIG. 7 is a linear correlation plot of hemacytometer counts that havebeen corrected for viability using methylene blue stain vs. Easy CountReadings. Data points are the mean of three samples. The linearrelationship is significant P<0.0001.

FIG. 8 is a linear correlation between percent viability as measured byslide culture, and Easy Count values. Data points are the mean of threesamples. The linear relationship is significant P<0.0001.

FIG. 9 is a plot representing fermentation tracking Fermentationtracking in a laboratory fermentation using both the Easy Count andhemacytometer methods. Time 0 is the time that the cells were pitchedinto fresh wort. The Y-axes represent cell counts using a hemacytometer(squares) and active cells using the Easy Count (diamonds). Data pointsare the mean of three samples.

FIG. 10 is a plot representing fermentation tracking during breweryscale fermentation. The Y-axes represent cell counts using ahemacytometer (squares) and the Easy Count (diamonds). Data points arethe mean of three samples.

FIG. 11 is a plot depicting Percent error between operators for thethree methods shown. Methylene Blue dead (stained) cells reporteddifferences between operators of 28.3%, while Methylene Blue live(non-stained cells) was 21.0%. The error between operators for the EasyCount was significantly lower, at only 2.6%.

DETAILED DESCRIPTION

Briefly, the current invention describes novel methods that can be usedto quantify live cells and total cells (total includes all cells in thesample, both viable and nonviable) such as yeast and bacteria. Thisallows the user to determine percent viability of the sample of cells.In one aspect, the instant invention comprises three steps: 1)determination of total cells, 2) determination of viable cells and 3)calculation of percent viability. In certain embodiments the total cellsare determined by washing and incubating the cells in a solution andthen measuring the native UV fluorescence of the cells in a fluorometer,thereby permitting the determination of total cell populations.Subsequently, the cells are incubated with a compound that can bemetabolically converted to a visible fluorescent dye such as fluoresceindiacetate, coupled with an inducer of esterase activity such asdequalinium acetate, and then the fluorescence is measured, thuspermitting enumeration of viable cell populations. The fluorescentreadings are correlated to standard counts such as hemacytometer countsor to the slide viability counts. The two fluorescence readings aredirectly related to the number of total and viable cells respectively.This permits the user to calculate the percent viability of a mixedpopulation of live and dead cells.

As those of ordinary skill in the art can readily appreciate the presentinvention may be modified in certain ways to achieve the same result. Inbrief, the present invention utilizes one or more dyes or molecules thatallow for the detection of all cells or total cells (e.g., yeast,bacteria, mammalian, etc.) in a sample and the same or different one ormore dyes that are metabolized/derivatized by the viable cells in thesample to allow detection of the viable cells. Accordingly, the percentviability can then be readily determined. As can be appreciated,substances such as detergent-like compounds, surfactants, solvents, orother compounds that affect membrane polarity, membrane fluidity,permeability, potential gradient, etc., may be added to the sample toincrease the rate at which the molecule or dye enters the cells in orderto speed the process.

Other variations that are within the scope of the present inventioninclude adding compounds that affect membrane polarity to decrease therate of “leakage” of the converted dye from the cells. Further, esteraseenzyme inducing chemicals such as naphthalene or dequalinium acetate maybe added to increase esterase activity in living cells. In addition,esterase activity may be increased by environmental factors such asheat. Furthermore, compounds other than fluorescein diacetate, such asCalcein AM, may also be used to detect metabolically active live cells.

The stability and shelf life of the fluorescein diacetate and otherchemicals may be increased by the addition of antioxidants or similarpreservatives or by dissolving the FDA into other solvents besidesacetone or by other stabilizing methods such as lyophilization. Thefluorescence detection apparatus used may be designed for microscopic,surface, internal, solution and non-suspension sample formats.

Also included within the context of the present invention is softwarethat permits the user to interface the fluorescence instrumentation to acomputer for direct calculation of percent viability and cellconcentration or other data processing or recording formats.

Compounds such as hemoglobin may be utilized to reduce background in thesample. Rinsing the sample in a buffer solution and centrifuging orfiltering or otherwise retaining the cells as they are washed can beused to remove any exogenous background fluorescence.

The differences between prokaryotes and eukaryotes may also utilized toassist detection. For example, such easily detectable differencesinclude cell membrane receptors, lack of organelles in prokaryotes, ormetabolic differences. These differences can be utilized to distinguishbetween prokaryotes and eukaryotes by using dyes that penetrate onlymitochondria or nuclei for example, or to take advantage of membrane andmetabolic differences in these two cell types. This will allow the userto count a specific prokaryote or a eukaryote in a mixture of cells thatcontains both types of cells. For example, one may determine if bacteriacontaminate a yeast cell or blood cell population.

Other variations of the present invention include altering the pH of thereaction solutions to increase the sensitivity of the reaction. Furthervariations include changing concentrations of the solutes to increase ordecrease the sensitivity of the reaction. Alterations to the solidstandards may be utilized to increase the sensitivity and dynamic rangeof the assay. The viability assay may be used to measure overall healthor metabolic or growth status of the cells, including the ability towithstand stress. The various steps of the tests may be usedindependently, e.g., to measure only total cells or only live cells.

Other methods of total cell determination may include using DNA bindingdyes, protein stains, cell membrane stains, antibody coupled stains,lipid dyes or other methods of detecting total cells in a sample. Viablecells may also be quantified using other methods that distinguishbetween live and dead cells such as surface markers, DNA stains, proteinstains, antibody coupled stains, lipid dyes or other methods ofdetecting viable cells in a sample.

The solid standard can be made out of other materials such as, but notlimited to, plastic, or by embedding chemicals such as fluorescein in asolid matrix of epoxy, acrylic, polyacrylamide or agarose or by coatinga material like plastic with said chemical. Other chemicals, which haveexcitation and emission wavelengths in the range of the dyes or cellsused to carry out the invention, could be used.

The instrument can be calibrated with solutions containing fluorescentchemicals such as fluorescein or OREGON GREEN™. Other configurations ofthe solid standard can be employed. The adaptor can be constructed tofit the type and make of instrument used to carry out the method.Different concentrations of the reagents may be used to carry out themethod. Other methods of mixing and or concentrating samples may beused. The wash steps may also be eliminated, thus simplifying theprocedure.

Yet further variations of the present invention include, but are notlimited to, the use of an incubator to control the temperature of thedye conversion in the cells. Such incubation can take place in a platecounter or vial heater of some kind Further, the samples may be arrangedin an array format to allow high throughput detection.

The methods and kits of the present invention allow for thedetermination of the number of active cells by measuring the rate ofconversion of dye by the cells. The number and activity of said cellsmay be determined without reaching the reaction endpoint.

The methodology has obvious application in determining the activity ofyeast or bacteria in industrial fermentation applications. Thus, themethods and kits can be used to predict the number of cells required tocarry out fermentation based on viable cells rather than on total cells.

As those of ordinary skill in the art can readily appreciate, theinstant invention can be carried out in a single vessel and solution.

The instant invention also has applicability in assessing the activity,vitality, or number of cells under various storage conditions, comparingthe metabolic activity of different cells, developing pitching ratecharts for fermentation applications, and use of the method as aself-contained laboratory.

The present invention provides methods, kits and apparatuses for simpledye associated quantitation that allows one to inexpensively determinetotal cell counts and viable cell counts in a particular sample. Anindividual of ordinary skill in the art will readily appreciate thatalternatives to the steps herein described for quantitating cells may beused and are encompassed herein. Accordingly, all alternatives will usea kit or method wherein a dye is utilized to stain cells and a method isused to detect or quantify the dye. One key aspect of this invention isits ability to simultaneously determine total cells and live cells in asample in short times compared to standard methods. In preferredembodiments, detection is completed in less than 4 hours, in others inless then about 3 hours, and yet further, in less than about 2 hours,while in specific embodiments, detection is completed in less than about1 hour, less than about 45 minutes, less than about 30 minutes, lessthan about 15 minutes, and less than about 10 minutes at low cost.

All patents, patent applications and references cited herein areincorporated in their entirety. Accordingly, incorporated herein byreference are U.S. Pat. Nos. 5,437,980; 5,563,070; 5,582,984; 5,658,751;5,436,134; 5,939,282; 4,783,401; 3,586,859.

EXAMPLES Example I Materials and Calibration

The following shows examples of solutions, volumes and concentrationsthat can be used to carry out the invention. Other concentrations andvolumes and different buffers with different pH values may be used. Thesamples and reaction solutions may be provided in any volume necessaryfor detection, however, in the present embodiment, small volumes (lessthan 1 ml) are utilized such that reagents and sample amounts are keptto a minimum. The volumes and concentrations to be utilized are any thatare convenient to the practitioner of the methodology. In certainembodiments, the sample and reagent amounts range from 1 to 10,000 microliters.

The reader used in the present embodiment is a “picofluor” hand-heldfluorometer from Turner Designs, Sunnyvale, Calif. 94086. Anyfluorometer that can measure fluorescence at specific wavelengths fordetection of the dyes or cells can be employed in the invention.Ideally, the fluorometer can switch back and forth from visible (486 nmexcitation with a 10 nm bandwidth and 550 emission with a 10 nmbandwidth) and UV (300-400 nm excitation and 410-700 nm emission) modeswithout changing filters or making other adjustments; however, this isnot necessary to carry out the methodology. Other wavelengths may beused in combination with different dye types or cell types. Thewavelength chosen will be dependent on the dye or cell type chosen forstaining.

The instrument needs to be calibrated in a range that permitsquantitation of cells. Calibration can be carried out with solutionssuch as fluorescein or OREGON GREEN™ or with the use of solid standardsas described below.

Materials Solid Calibration Standards:

Materials

-   -   Colored glass rods: Mint green for the visible mode calibration,        and translucent blue for the ultraviolet (UV) mode calibration.    -   Solid calibration adapter: Black diacetyl plastic machined to        fit the instrument. Glass rods are glued into the solid        calibration adapter and sealed with a plastic cap, (See FIG. 1).

Solutions A (Cell Preparation): 1×PBS

-   -   Ingredients (for 10× stock solution)    -   80 grams NaCl    -   2.0 grams KCl    -   14.4 grams Na₂HPO₄    -   2.4 grams KH₂PO₄    -   NaOH (enough to reach a PH of 7.4)    -   This recipe makes a 10× stock solution. It must be diluted 1:10        into distilled water to make a 1× working solution before being        used.

Solution B (Live Cell Suspension):

-   -   Ingredients: 0.0275 grams dequalinium acetate in    -   30 mL 10× Sodium acetate buffer: (13.6 grams Sodium acetate in        100 mL ddH₂o)

Stock Solution C (Live Cell Reaction Stock):

-   -   Ingredients:    -   Fluorescein diacetate (FDA), 30 mg/10 mL in Acetone

Protocol

I. Calibration Using Solid Standards

To calibrate the instrument in “A” mode (UV): Standard Value=500

-   -   1) Remove the mini cell receptacle from the instrument.    -   2) Be sure that the instrument is in “A” mode; the letters UV        should appear in the lower left corner of the screen.    -   3) If the instrument is not in “A” mode, press the A/B key on        the instrument keypad.    -   4) Press the CAL key on the keypad; when prompted, press ENTER        to continue.    -   5) When asked to insert the blank, insert the solid standard        labeled “A” into the instrument with the letter “A” facing down        and to the right.    -   6) When asked to insert the “cal”, insert the solid standard        labeled “A” into the instrument so that the letter A is facing        towards you, and the white cap is on top.

To calibrate the instrument in “B” mode (Visible): Standard Value=500

-   -   1) Follow all the instructions for calibration in “A” mode,        making sure that that the instrument is now in “B” mode, and        that the solid standard labeled “B” is now being used.

Example II Total Cell Counts

Total cell counts can be determined by a variety of methodologies,including using the fluorometer noted above. UV operation mode isselected. A fixed volume (200 microliters) of Cell preparation solutionis added to the glass sample vial. 5 microliters of sample (yeast cells)is then added to the Cell preparation solution in the vial andcentrifuged for about 30 seconds to sediment the cell pellet. The cellpellet is resuspended in about 100 microliters of Cell preparationsolution to the sample vial. The sample vial is then read influorometer. See FIG. 2.

Example III Viable Cell Counts

Live cells are quantitated utilizing a dye that is detectably altered byan intracellular enzyme. For example, the fluorometer noted above is setin visible mode and a fixed volume (200 microliters) of Cell preparationsolution is added to the sample vial. 5 microliters of sample is addedto the solution A in the sample vial and centrifuged for 30 seconds. 100microliters of Suspension solution is added to the sample vial and 5microliters of Reaction solution is added to the sample vial. The sampleis mixed and placed in the reader and fluorescence determined at timezero and again at 15 minutes. This is the value that will be compared tothe Easy Count correlation chart for conversion to cells/ml. See FIG. 3.

Example IV Yeast Quantitation

Yeast performance is critical to the development of quality beer. Forthis reason, methods of yeast analysis are an important element of thebrewing process. Traditional methods including hemacytometer countingand methylene blue staining are rapid, but inaccurate and unreliable.Slide culture is an accurate measure of yeast viability, but requires alengthy incubation period of 18 to 24 hours. As an alternative, thefluorometric assay described above is based on the metabolic activity ofthe yeast culture to provide brewers with a rapid and accurateestimation of active cell number. This method was compared to thehemacytometer counting technique as an estimation of cell number, and toboth methylene blue staining and slide culture as measures of vitalityand prediction of fermentation performance. The inventive methodcorrelated to the hemacytometer, methylene blue, and slide culture withR² values of 0.985, 0.987, and 0.962 respectively, P<0.0001. An erroranalysis was carried out by the inventive methods, hemacytometer andmethylene blue staining techniques for multiple operators performing thetests. Thus, the present invention could be used to determine correctpitching rates, monitor fermentation and propagation, and for otherapplications involving cell quantitation.

Yeast

All yeast cultures were obtained from Wyeast Laboratories, Mt. Hood,Oreg. Yeast samples for the experiments comparing the hemacytometer,methylene blue staining, and slide culture to the inventive methods werea 1084 strain of Saccharomyces cerevisiae. Yeast cultures tested duringlaboratory scale fermentations were strain 1968. Brewery scalefermentations were performed using yeast strain 1056.

Hemacytometer Counts

Hemacytometer counts were performed according to the ASBC method (6,8).Samples were removed from a slurry with an initial concentration of 198million cells per ml, as determined by hemacytometer count, and dilutedin spent wort to maintain cell integrity. Each dilution was counted inthe hemacytometer as well as measured using the present method.Experiments were carried out in triplicate.

Methylene Blue Staining

Methylene blue staining was performed according to the ASBC method(6,8). Samples were removed from a slurry with an initial concentrationof 198 million cells per ml, as determined by hemacytometer count, anddiluted in spent wort to maintain cell integrity. Each dilution wasstained and counted in a hemacytometer, as well as measured using thepresent method. Hemacytometer counts were corrected for viabilityaccording to the staining results. Experiments were carried out intriplicate.

Slide Culture

Slide culture was performed according to a modified version of theprotocol for preparation of slide cultures for the examination of yeastand mold (5). Ten ml of yeast strain 1028 at a concentration of 433million cells per milliliter, as determined by a hemacytometer count,were placed into a 43° C. water bath. Aliquots were removed at timeintervals 0, 2, 4, 6, 8, 10, 15, 20, 30, and 40 minutes. Each was testedusing the inventive method. Measurements were taken in triplicate andaveraged. Slide culture samples were diluted 1:100 into wort containing6% gelatin. 10 μl of the sample were then placed on a micro slide,covered and sealed with petroleum jelly. Each slide was incubated for 20hours at 18° C. before microscopic examination (Microscope model, LeicaDMLB). Viability was determined with the assumption that living cellshad formed micro colonies, while nonviable cells remained single.

Determination of Active Cell Number Using the Instant Invention

The inventive method for determining total active cell number is basedon the metabolic activity of the yeast culture. The technology involvesexposing cells to proprietary chemicals that enter cells throughdiffusion. These molecules are converted to a fluorescent form bymetabolically active cells. This fluorescent signal is quantified in ahandheld battery operated fluorometer model GP320 GenPrime Inc, Spokane,Wash. The protocol is as follows: 50 μl of yeast sample was added to 500μl of cell prep solution in a 1 ml glass test cuvette. 50 μl of dyesolution was added; the cuvette was capped, and incubated for 5 minutes.After incubation, the cuvette was shaken, and the fluorescent signalquantitated in the GP320. These values were compared to thehemacytometer and methylene blue staining methods by performing testswith the inventive method on the diluted samples from these experiments.Readings were taken in triplicate and averaged. These relationships wereanalyzed by linear regression using Statview, SAS institute, Cary N.C.

Fermentation Tracking

Laboratory Scale: Laboratory scale fermentation tracking was carried outin a 300 ml flask by inoculating 150 ml wort with 5 ml yeast strain1968, with an initial concentration of 420 million cells per ml, andmonitoring growth using a hemacytometer and the inventive methods. Cellswere grown at room temperature (21° C.). Samples were taken every 45minutes for 5.25 hours and then periodically over the next 48 hours.

Brewery Scale Brewery Scale fermentation tracking was carried out duringa typical fermentation cycle, at the Steam Plant Grill, Spokane, Wash.99201. Hemacytometer counts and corresponding readings using the instantinvention were made daily for 13 days beginning immediately followingpitching.

Error

Percent error between operators was determined for the inventive method“Easy Count” method, hemacytometer counts, and the methylene bluestaining method. Error analysis was performed using Microsoft Excel.

Hemacytometer: Three operators performed hemacytometer analysis of ayeast strain 1028 slurry according to the ASBC method. Each operatorprepared and measured 15 samples. Results were averaged for eachoperator, and error between operators was calculated.

Methylene Blue: The 15 hemacytometer samples from above were stainedwith methylene blue according to the ASBC method. Each of the threeoperators counted stained cells for each sample. Results were averagedfor each operator, and error between operators was calculated.

Easy Count: Easy Count tests were performed on 15 replicate samples byeach of the three operators. Results were averaged for each operator,and error between operators was calculated.

Hemacytometer Counts

FIG. 6 shows the correlation between the Easy Count values and thecells/ml results of the hemacytometer. A statistically linearrelationship was found between cell counts obtained by the ASBC standardmethod of microscopic examination using a hemacytometer and valuesobtained using the Easy Count, R²=0.985.

Methylene Blue Staining

FIG. 7 illustrates the linear correlation found between the Easy Countmethod and the ASBC method for methylene blue staining A statisticallylinear relationship was found between the Easy Count, and hemacytometercounts corrected for viability, R²=0.987

These results suggest that the Easy Count can be used to accuratelypredict active cell number. Using the results of the correlation, it ispossible for the brewer to accurately determine the correct pitchingrate using the Easy Count method based on 1 million active cells per mlper degree plato of wort. Additionally, the method can be used tomonitor fermentation, propagation, and for other applications involvingthe quantitation of cells.

Slide Culture

A linear relationship was found between the Easy Count and slide culturefor yeast viability, as shown in FIG. 8.

The correlation to slide culture confirms that the Easy Count onlymeasures active cells, since the total number of cells in thisexperiment remains constant.

Fermentation Tracking

Cell growth was measured during laboratory and brewery scalefermentations using both the ASBC method for hemacytometer counts, andthe Easy Count method. FIG. 9 shows cell growth tracked by both methodsduring laboratory scale fermentation. FIG. 10 is an example of a breweryscale fermentation tracked by both methods.

Error

Results from the experiments were averaged for each operator as shown inTable 1. Percent error between operators was calculated by dividing thestandard deviation of the mean by the mean, and multiplying the resultby 100. Easy Count reported significantly lower error between operatorsthan the other methods. These results are graphed in FIG. 11.

TABLE I Data in Easy Methylene Methylene Millions of Count Blue liveBlue dead cells/ml mean mean mean Operator 1 195.9 158.6 27.9 Operator 2197 122.3 23.6 Operator 3 187.7 187.7 40.3 Mean 193.5 156.2 30.6 Std.Dev. 5.1 32.8 8.7 % Error 2.6 21.0 28.3

Results of the error experiments confirm previous research reporting theinaccuracies of hemacytometer counts and methylene blue staining (1, 2,3, 4, 7). The low error associated with the Easy Count method is animprovement on these traditional techniques.

Percent error is of particular importance to the brewer due to theexacerbation of inaccuracies in the calculation of cells/ml. Forexample, when calculating cells/ml from a hemacytometer count of 180live cells and 15 dead cells (counting all 25 fields and using a 1:100dilution), the result would be 180 million live cells/ml (180*100*10000)and 15 million dead cells/ml (15*100*10000). If the error betweenoperators when performing the live cell test is 21%, then the live cellresult could be between 142-218 million cells/ml, a difference of 76million cells/ml. With a percent error of 28% between operators, thedead cell result could be between 11-19 million cells/ml. This couldresult in reported viabilities between 87% and 96% for the same sample.The Easy Count has much less error associated with its performance. Areading of 6000 in the Easy Count would be 197 million active cells/ml(see equation generated in FIG. 7.). A percent error of 3% betweenoperators gives a range between 191-203 million cells/ml, a differenceof only 12 million cells/ml. The very low error associated with theperformance of the Easy Count provides much more reliable information tothe brewer.

-   1. Mochaba, F. et al, Practical Procedures to Measure Yeast    Viability and Vitality Prior to Pitching. J. Am. Soc. Brew. Chem.    56(1): 1-6, 1998.-   2. O'Connor-Cox, E. et al, Methylene Blue Staining: use at your own    risk. Tech. Q. Master. Brew. Assoc. 34:306-312, 1997-   3. Carvell J. P. et at Developments in Using Off-Line Radio    Frequency Impedance Methods for Measuring the Viable Cell    Concentration in the Brewery. J. Am. Soc. Brew. Chem. 58(2): 57-62,    2000-   4. Smart, K. A. et at Use of Methylene Violet Staining Procedures to    Determine Yeast Viability and Vitality. J. Am. Soc. Brew. Chem.    57(1): 18-23, 19992.-   5. Harrigan, W. F. Laboratory Methods in Food Microbiology 3^(rd)    Ed. Academic Press, San Diego, Calif. 1998-   6. American Society of Brewing Chemists. Methods of Analysis, 8^(th)    Ed. The society, St. Paul. MN. 1992.-   7. Koch, H. A., et al, Fluorescence Microscopy Procedures for    Quantitation of Yeasts in Beverages. American Society for    Microbiology, 52(3): 599-601, September 1986.-   8. Allen, P. The Microbrewery Laboratory Manual—A Practical Guide to    Laboratory Techniques and Quality Control Procedures for Small Scale    Brewers, Part 1: Yeast Management. Brewing Techniques 2(4): 28-35    July/August, 1994.

Other References

-   1. Catt, S. L. Sakkas, D., Bizarro, D. Bianchi, P. G.,    Maxwell, W. M. and Evans, G.:-   (1977) Molecular and Human Reproduction 3: 821-825.-   2. Ferguson, L. R. and Denny, W. A.: (1995) Mutation Research 329:    19-27.-   3. Latt, S. A. and Wohleb, J. D. (1975) Chromosoma 52:297-316.-   4. Harrington, W. F., (1998) Laboratory Methods in food    Microbiology, Academic Press, San Diego, Calif.-   5. Brailsford, M. A. and Gatley, S. (1993) New Techniques in Food    and Beverage Microbiology (ed. R. G. Kroll, A Gilmour and M.    Sussman), Oxford : Blackwell Scientific.-   6. Griffith, C. J. Blucher, A. and Fleri, J. (1994) Food Science and    Technology today 8: 209-216-   7. Pettipher, G. L. Krollo, R. G. and Fan, L. J. (1989) Rapid    Microbiological Methods for Foods, Beverages and Pharmaceuticals    (ed C. J. Stannard, S. B. Pettit and F A Skinner) Oxford: Blackwell    Scientific.-   8. Pinder, A. C. Edwards, C., and Clarke, R. G. (1993) New    Techniques in Food and Beverage Microbiology (ed. R. G. Kroll, A    Gilmour and M. Sussman), Oxford : Blackwell Scientific.-   9. Stannard, C. J. Pettit, S. B. and Skinner, F. A. (1989) Rapid    Microbiological Methods for Foods, Beverages and Pharmaceuticals    (ed C. J. Stannard, S. B. Pettit and F A Skinner) Oxford: Blackwell    Scientific.-   10. Catt, S. L. Sakkas, D., Bizarro, D. Bianchi, P. G.,    Maxwell, W. M. and Evans, G.: (1977) Molecular and Human    Reproduction 3: 821-825.-   11. Ferguson, L. R. and Denny, W. A. (1995) Mutation Research 329:    19-27.-   12. Atlas, R. M. and Bartha, R. Microbial Ecology, Addison Wesley,    Longman, New York, (1998).-   13. Guldfeldt, L. U. Arneborg, N. Siegumfeldt, H. and Jespersen, L.    Relationship between yeast cell proliferation and intracellular    esterase activity during brewing fermentations. J. Inst. Brew.    333-338.-   14. Breeuwer, P. et al. (1995). Characterization of uptake and    hydrolysis of fluorescein diacetate and carboxyfluorescein diacetate    by intracellular esterases in saccharomyces cerevesiae, which result    in accumulation of fluorescent product. Applied and Environmental    Microbiology, 61: 1614-1619.-   15. Prosperi, E. (1990) Intracellular turnover of fluorescein    diacetate. Influence of membrane ionic gradients on fluorescein    efflux. Histochemical Journal 22: 227-233.-   16. Breeuwer, P. Drocourt, J., Rombouts, F. M. and Abee, T. (1994)    Energy-dependent, carrier-mediated extrusion of carboxyfluorescein    from saccharomyces cerevesiae allows rapid assessment of cell    viability by flow cytometry. Applied and Environmental Microbiology,    1467-1472.

1.-38. (canceled)
 39. A method for detecting viable yeast or theactivity thereof in industrial fermentation applications, comprising:(a) providing a sample containing yeast cells, (b) contacting saidsample containing yeast cells with fluorescein diacetate or OREGONGREEN™ (2′,7′ difluorofluorescein), (c) detecting fluorescence of saidsample, wherein the detection is performed by a fluorometer; (d)repeating step (c) over a time course, wherein a change in fluorescenceover the time course indicates a change in activity or number of viableyeast cells.
 40. The method of claim 39, wherein an increase influorescence indicates increased metabolic activity or an increase inviable yeast cell number.
 41. The method of claim 39, wherein a decreasein fluorescence indicates a decrease in metabolic activity or a decreasein viable yeast cell number.
 42. The method of claim 39, wherein theyeast is Sacchromyces cerevisiae.
 43. The method of claim 39, whereinstep (c) further comprises rinsing the sample in a buffer solution andcentrifuging or filtering or otherwise retaining the yeast cells as theyare washed to remove any exogenous background fluorescence prior to thedetection of fluorescence.
 44. The method of claim 39, wherein the yeastcells in the sample are concentrated prior to step (b).
 45. The methodof claim 39, wherein step (b) further comprises contacting the yeastcells with a compound that increases the rate of uptake of dye into theyeast cells or speeds up the rate of conversion the detectablefluorescent form of the dye inside said yeast cells.